Process for the deacylation of cyclic lipopeptides

ABSTRACT

There is provided a cyclic lipopeptide acylase which is capable of deacylating the acyl side chain of a cyclic lipopeptide compound, e.g. Substance FR901379 or its analog, of the following general formula [I] with effectiveness as well a method of producing a cyclic peptide compound which comprises using the acylase.                    
     [wherein R 1  is acyl; 
     R 2  is hydroxy or acyloxy; 
     R 3  is hydrogen or hydroxy; 
     R 4  is hydrogen or hydroxy; 
     R 5  is hydrogen or hydroxysulfonyloxy; and 
     R 6  is hydrogen or carbamoyl.]

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. Ser. No.09/142,045 filed on Sep. 3, 1998, now U.S. Pat. No. 6,207,434, which wasoriginally filed as International Application No. PCT/JP97/00692 on Mar.6 1997.

TECHNICAL FIELD

The present invention relates to enzyme technology.

The present invention relates to a novel acylase capable of deacylatingthe acyl side chain of a cyclic lipopeptide compound and to adeacylation method using the same.

More particularly, the invention relates to a novel acylase adapted todeacylate the acyl side chain of Substance FR901379 (described inJapanese Kokai Tokkyo Koho H3-184921), which is produced by themicroorganism Coleophoma sp. F-11899 (FERM BP-2635), or an analog ofSubstance FR901379 and to a deacylation method using the same.

BACKGROUND ART

There has been a standing need for an acylase capable of deacylating theacyl side chain of a cyclic lipopeptide compound, specifically saidSubstance FR901379 or analog, with good efficiency.

DISCLOSURE OF THE INVENTION

The inventors of the present invention did an extensive research insearch of a novel acylase capable of deacylating the acyl side chain ofa cyclic lipopeptide represented by Substance FR901379 or its analogssuch as Echinocandin B and Aculeacin A. As a result, they discovered anacylase produced by Streptomyces anulatus and succeeded in effectiveachievement of the objective deacylation.

The above novel cyclic lipopeptide acylase and the deacylation methodusing the same are now described with reference to their salientfeatures.

First, the cyclic lipopeptide acylase-producing microorganism isdescribed.

The novel cyclic lipopeptide acylase-producing microorganism includesbut is not limited to Streptomyces anulatus No. 4811, Streptomycesanulatus No. 8703, and Streptomyces sp. 6907, all of which belong to thegenus Streptomyces.

The characteristics of those strains are now described.

The novel cyclic lipopeptide acylase-producing strain named Streptomycesanulatus No. 4811 (herein after referred to briefly as Strain No. 4811)was isolated for the first time from a soil sample collected inFukushima Prefecture. The bacteriological characteristics of this StrainNo. 4811 are now described.

Cultural Characteristics

The cultural characteristics of Strain No. 4811 on yeast extract-maltextract agar, oatmeal agar, inorganic salts-starch agar,glycerin-asparagine agar, peptone-yeast extract-iron agar, and tyrosineagar after incubation at 30° C. for 14 days and the light and scanningelectron microscopic observation of the respective growths aresummarized in Table 1. The color descriptions given below correspond tothe nomenclature defined in Methuen Handbook of Colour, Methuen, London,1978.

TABLE 1 Cultural characteristics of Strain NO. 4811 Medium Culturalcharacteristics Yeast extract- G: good malt extract agar A: abundant,yellowish gray (2B2) (ISP-2) R: brown (7F4) S: scanty, brown Oatmealagar G: good-moderate (ISP-3) A: abundant, yellowish gray (2C3) R: brown(7F4) S: trace, brown Inorganic salts- G: good Starch agar A: abundant,yellowish gray (2C3) (ISP-4) R: yellowish brown (5E4) S: noneGlycerin-asparagine agar G: good (ISP-5) A: abundant, yellowish gray(2C3) R: brown (6E4) S: none Peptone-yeast extract-iron G: moderate agarA: poor, white (ISP-6) R: light brown (6D5) S: none Tyrosine agar G:moderate (ISP-7) A: moderate, yellowish gray (2B2) R: brown (7F4) S:none Codes: G: growth, A: aerial mycelium, R: reverse side color, S:soluble pigment

The color of the aerial mycelium was yellowish gray to greenish gray,the reverse side color of growth was yellowish brown to brown, thesoluble pigment was light brown, and neither intracellular pigments norsoluble pigments were pH-sensitive. No melanoid pigments were produced.

Physiological Characteristics

The physiological characteristics of Strain No. 4811 are summarized inTable 2.

TABLE 2 Physiological characteristics of Strain No. 4811 Test itemResult Temperature range for growth 4.0-35.0° C. Liquefaction ofgelatin + Coagulation of milk ± Peptonization of milk + Hydrolysis ofstarch + Production of Melanoid pigments − Carbon utilization:D-glucose + L-arabinose + D-xylose + Inositol − Mannitol + D-fructose +L-rhamnose ± Sucrose − Raffinose − +: positive, ±: weakly positive, −:negative

The vegetative mycelium of Strain No. 4811 developed well and branchedirregularly but not fragmented. The aerial mycelium extending from thevegetative mycelium branched monopodially to form elongated sporechains. The spore chain morphology of the aerial mycelium wasstraight-flexuous, thus belonging to the RF type according to theclassification of Pridham et al. (Pridham, T. G. et al.: Appl.Microbiol., 6:54, 1958). Each spore chain consisted of 20 or morespores. The spores were smooth-surfaced (glabrous) and cylindrical. Thespore size was 0.5˜0.7×0.7˜1.1 μm.

None of sclerotium, sporangium, and zoospore was observed.

Cell Wall Type

As to the cell wall amino acid composition, the whole-cell lysate wasanalyzed by the method of Becker et al. (Becker, B., M. P. Lechevalier,R. E. Gordon and H. A. Lechevalier: Rapid differentiation betweenNocardia and Streptomyces by paper chromatography of whole-cellhydrolysates: Appl. Microbiol., 12:421-423, 1964)and the method ofYamaguchi (Yamaguchi, T.: Comparison of the cell wall composition ofmorphologically distinct actinomycetes: J. Bacteriol., 89:444-453,1965). The result indicated the existence of LL-diaminopimelic acid.Therefore, the cell wall of this strain was considered to be of Type I.

Based on the above morphological observation and chemical analysis,Strain No. 4811 was considered to belong to the genus Streptomycesaccording to the taxonomic classification of Pridham et al. (Pridham, T.G. et al: Appl. Microbiol., 6:54, 1958). Accordingly, thecharacteristics of this strain were compared with those of Streptomycesspecies as described in the literature, namely Shirling et al.(Shirling, E. B. and D. Gottlieb: Cooperative Description of TypeCulture of Streptomyces. 2. Species descriptions from first study,Intern. J. Syst. Bacteriol., 18:69-189, 1968; Shirling, E. B. and D.Gottlieb: Cooperative Description of Type Culture of Streptomyces. 3.Additional species descriptions from first and second studies, Intern.J. Syst. Bacteriol., 18:279-392, 1968; Shirling, E. B. and D. Gottlieb:Cooperative Description of Type Culture of Streptomyces. 4. Speciesdescriptions from second, third and forth studies, Intern. J. Syst.Bacteriol., 19:391-512, 1969); Skerman et al. (Skerman, V. B., V.McGowan and P. H. A. Sneath: Approved List of Bacterial Names, AmendedEdition, American Society for Microbiology, Washington D.C., 1989); andMoore et al. (Moore, W. E., E. P. Cato and L. V. H. Moore: Index ofBacterial and Yeast Nomencultural Changes, American Society forMicrobiology, Washington D.C., 1992). The comparison indicated that thecharacteristics of Streptomyces anulatus so described were substantiallyidentical to the characteristics of this strain. Accordingly, thisStrain No. 4811 was identified as Streptomyces anulatus and namedStreptomyces anulatus No. 4811.

This Streptomyces anulatus No. 4811 was originally deposited withNational Institute of Bioscience and Human Technology (NIBH, 1-3,Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan) (ZIP code 305) on Dec. 27,1995 under the accession number of FERM P-15377 and subsequentlyconverted to deposit according to the Budapest Treaty as of Feb. 3, 1997under the accession number of FERM BP-5808.

The novel cyclic lipopeptide acylase-producing strain named Streptomycesanulatus No. 8703 (herein after referred to briefly as Strain No. 8703)was isolated for the first time from a soil sample collected inFukushima Prefecture. The bacteriological characteristics of this StrainNo. 8703 are now described.

Cultural Characteristics

The cultural characteristics of Strain No. 8703 on yeast extract-maltextract agar, oatmeal agar, inorganic salts-starch agar,glycerin-asparagine agar, peptone-yeast extract-iron agar, and tyrosineagar after incubation at 30° C. for 14 days and the light and scanningelectron microscopic observations of the respective growths aresummarized in Table 3. The color descriptions given below correspond tothe nomenclature defined in Methuen Handbook of Colour, Methuen, London,1978.

TABLE 3 Cultural characteristics of Strain No. 8703 Medium Culturalcharacteristics Yeast extract- G: good malt extract agar A: abundant,yellowish gray (2B2) (ISP-2) R: gray brown (5F4) S: none Oatmeal agar G:moderate (ISP-3) A: abundant, yellowish gray (2C3) R: gray brown (4C4)S: none Inorganic salts- G: good starch agar A: abundant, yellowish gray(2C3) (ISP-4) R: dark gray (1F6) S: none Glycerin-asparagine agar G:good (ISP-5) A: abundant, yellowish gray (2C3) R: olive brown (4E5) S:none Peptone-yeast extract-iron G: moderate agar A: poor, white (ISP-6)R: yellowish brown (5D5) S: none Tyrosine agar G: moderate (ISP-7) A:moderate, yellowish gray (2B2) R: brown (7F4) S: none Codes: G: growth,A: aerial mycelium, R: reverse side color, S: soluble pigment

The color of the aerial mycelium was yellowish gray to greenish gray,the reverse side color of growth was yellowish brown to brown, thesoluble pigment was light brown, and neither intracellular pigments norsoluble pigments were pH-sensitive. Melanoid pigments were produced intryptone-yeast extract broth, peptone-yeast extract-iron agar, andtyrosine agar.

Physiological Characteristics

The physiological characteristics of Strain No. 8703 are summarized inTable 4.

TABLE 4 Physiological characteristics of Strain No. 8703 Test itemResult Temperature range for growth 4.0-35.0° C. Liquefaction ofgelatin + Coagulation of milk ± Peptonization of milk + Hydrolysis ofstarch + Production of Melanoid pigments − Carbon utilization:D-glucose + L-arabinose + D-xylose + Inositol − Mannitol + D-fructose +L-rhamnose + Sucrose − Raffinose − +: positive, ±: weakly positive, −:negative

This strain did not utilize inositol, sucrose, and raffinose. Itpeptonized milk. The temperature range for growth was 4.0˜35° C.

The vegetative mycelium of Strain No. 8703 was developed well andbranched irregularly but was not fragmented. The aerial myceliumextending from the vegetative mycelium branched monopodially to formelongated spore chains. The spore chain morphology of the aerialmycelium was straight-flexuous, thus, belonging to the RF type accordingto the classification of Pridham et al. (Pridham, T. G. et al.: Appl.Microbiol., 6:54, 1958). Each spore chain consisted of 20 or morespores. The spore was smooth-surfaced and cylindrical. The spore sizewas 0.5˜0.8×0.6˜1.1 μm. None of sclerotium, sporangium, and zoospore wasobserved.

Cell Wall Type

As to the cell wall amino acid composition, the whole-cell lysate wasanalyzed by the method of Becker et al. (Becker, B., M. P. Lechevalier,R. E. Gordon and H. A. Lechevalier: Rapid differentiation betweenNocardia and Streptomyces by paper chromatography of whole-cellhydrolysates: Appl. Microbiol., 12:421-423, 1964) and the method ofYamaguchi (Yamaguchi, T.: Comparison of the cell wall composition ofmorphologically distinct actinomycetes: J. Bacteriol., 89:444-453,1965). The analysis indicated the existence of LL-diaminopimelic acid.Therefore, the cell wall of this strain is considered to be of Type I.

Based on the above morphological observation and chemical analysis,Strain No. 8703 was considered to belong to the genus Streptomycesaccording to the taxonomic classification of Pridham et al. (Pridham, T.G. et al: Appl. Microbiol., 6:54, 1958). Accordingly, the characters ofthis strain were compared with those of Streptomyces species asdescribed in the literature, namely Shirling et al. (Shirling, E. B. andD. Gottlieb: Cooperative Description of Type Culture of Streptomyces. 2.Species descriptions from first study, Intern. J. Syst. Bacteriol.,18:69-189, 1968; Shirling, E. B. and D. Gottlieb: CooperativeDescription of Type Culture of Streptomyces. 3. Additional speciesdescriptions from first and second studies., Intern. J. Syst.Bacteriol., 18:279-392, 1968; Shirling, E. B. and D. Gottlieb:Cooperative Description of Type Culture of Streptomyces. 4. Speciesdescriptions from second, third and forth studies, Intern. J. Syst.Bacteriol., 19:391-512, 1969); Skerman et al. (Skerman, V. B., V.McGowan and P. H. A. Sneath: Approved List of Bacterial Names, AmendedEdition, American Society for Microbiology, Washington D.C., 1989); andMoore et al. (Moore, W. E., E. P. Cato and L. V. H. Moore: Index ofBacterial and Yeast Nomencultural Changes, American Society forMicrobiology, Washington D.C., 1992). The comparison indicated that thecharacteristics of Streptomyces anulatus so described were substantiallyidentical to the characteristics of this strain. Accordingly, thisStrain No. 8703 was identified as Streptomyces anulatus and namedStreptomyces anulatus No. 8703.

This Streptomyces anulatus No. 8703 was originally deposited withNational Institute of Bioscience and Human Technology (NIBH, 1-3,Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan) (ZIP code 305) on Mar. 8,1996 under the accession number of FERM P-15507 and subsequentlyconverted to deposit according to the Budapest Treaty as of Feb. 3, 1997under the accession number of FERM BP-5810.

The novel cyclic lipopeptide acylase-producing strain named Streptomycessp. No. 6907 (herein after referred to briefly as Strain No. 6907) wasisolated for the first time from a soil sample collected in FukushimaPrefecture. The bacteriological characteristics of this Strain No. 6907are now described.

Cultural Characteristics

The cultural characteristics of Strain No. 6907 on yeast extract-maltextract agar, oatmeal agar, inorganic salts-starch agar,glycerin-asparagine agar, peptone-yeast extract-iron agar, and tyrosineagar after incubation at 30° C. for 14 days and the light and scanningelectron microscopic findings of the respective growths are summarizedin Table 5. The color descriptions given below correspond to thenomenclature defined in Methuen Handbook of Colour, Methuen, London,1978.

TABLE 5 Cultural characteristics of Strain No. 6907 Medium Culturalcharacteristics Yeast extract- G: good malt extract agar A: abundant,yellowish gray (ISP-2) (white 4A2) R: grayish orange (5B6) S: noneOatmeal agar G: moderate (ISP-3) A: abundant, bluish gray (22C2) R:light brown (4D4) S: none Inorganic salts- G: good starch agar A:abundant, bluish gray (19C2) (ISP-4) R: brown (6F4) S: noneGlycerin-asparagine agar G: good (ISP-5) A: abundant, bluish gray (22B2)R: reddish brown (8E4) S: none Peptone-yeast extract-iron G: moderateagar A: none (ISP-6) R: grayish brown (9F3) S: dark brown Tyrosine agarG: good (ISP-7) A: moderate, yellowish white (4A2) R: dark magenta(13F3) S: dark brown Codes: G: growth, A: aerial mycelium, R: reverseside color, S: soluble pigment

The color of the aerial mycelium was yellowish gray to bluish gray, thereverse side color of growth was light brown to brown, and theintracellular pigments were not pH-sensitive. Melanoid pigments wereproduced in tryptone-yeast extract broth, peptone-yeast extract-ironagar, and tyrosine agar.

Physiological Characteristics

The physiological characteristics of Strain No. 6907 are summarized inTable 6.

TABLE 6 Physiological characteristics of Strain No. 6907 ParametersFindings Temperature range for growth 9.0-40.0° C. Liquefaction ofgelatin + Coagulation of milk + Peptonization of milk − Hydrolysis ofstarch + production of Melanoid pigments + Carbon utilization:D-glucose + L-arabinose + D-xylose + Inositol + Mannitol + D-fructose +L-rhamnose + Sucrose + Raffinose + +: positive, ±: weakly positive, −:negative

This strain utilized all the carbon sources tested. It did not peptonizemilk. The temperature range for growth was 9.0˜40.0° C.

The vegetative mycelium of Strain No. 6907 developed well and branchedirregularly but was not fragmented. The aerial mycelium extending fromthe vegetative mycelium branched monopodially to form elongated sporechains. The spore chain morphology of the aerial mycelium wasstraight-flexuous or incomplete loops, thus belonging to the RF or RAtype according to the classification of Pridham et al. (Pridham, T. G.et al.: Appl. Microbiol., 6:54, 1958). Each spore chain consists of 20or more spores. The spore is smooth-surfaced and cylindrical. The sporesize is 0.5˜0.7×0.7˜1.3 μm. None of sclerotium, sporangium, and zoosporewas observed.

Cell Wall Type

As to the cell wall amino acid composition, the whole-cell lysate wasanalyzed by the method of Becker et al. (Becker, B., M. P. Lechevalier,R. E. Gordon and H. A. Lechevalier: Rapid differentiation betweenNocardia and Streptomyces by paper chromatography of whole-cellhydrolysates: Appl. Microbiol., 12:421-423, 1964) and the method ofYamaguchi (Yamaguchi, T.: Comparison of the cell wall composition ofmorphologically distinct actinomycetes: J. Bacteriol., 89:444-453,1965). The analysis indicated the existence of LL-diaminopimelic acid.Therefore, the cell wall of this strain was considered to be of Type I.

Based on the above morphological observation and chemical analysis,Strain No. 6907 was considered to belong to the genus Streptomycesaccording to the taxonomic classification of Pridham et al. (Pridham, T.G. et al: Appl. Microbiol., 6:54, 1958). Accordingly, the characters ofthis strain were compared with those of Streptomyces species asdescribed in the literature, namely Shirling et al. (Shirling, E. B. andD. Gottlieb: Cooperative Description of Type Culture of Streptomyces. 2.Species descriptions from first study, Intern. J. Syst. Bacteriol.,18:69-189, 1968; Shirling, E. B. and D. Gottlieb: CooperativeDescription of Type Culture of Streptomyces. 3. Additional speciesdescriptions from first and second studies, Intern. J. Syst. Bacteriol.,18:279-392, 1968; Shirling, E. B. and D. Gottlieb: CooperativeDescription of Type Culture of Streptomyces. 4. Species descriptionsfrom second, third and forth studies, Intern. J. Syst. Bacteriol.,19:391-512, 1969); Skerman et al. (Skerman, V. B., V. McGowan and P. H.A. Sneath: Approved List of Bacterial Names, Amended Edition, AmericanSociety for Microbiology, Washington D.C., 1989); and Moore et al.(Moore, W. E., E. P. Cato and L. V. H. Moore: Index of Bacterial andYeast Nomencultural Changes, American Society for Microbiology,Washington D.C., 1992). The comparison failed to indicate a species towhich the strain could be identified and, therefore, this strain wasnamed Streptomyces sp. No. 6907.

This Streptomyces sp. No. 6907 was originally deposited with NationalInstitute of Bioscience and Human Technology (NIBH, 1-3, Higashi1-chome, Tsukuba-shi, Ibaraki, Japan) (ZIP code 305) on Mar. 8, 1996under the accession number of FERM P-15506 and subsequently converted todeposit according to the Budapest Treaty as of Feb. 3, 1997 under theaccession number of FERM BP-5809.

The term “cyclic lipopeptide compound” as used in this specificationmeans a compound having a polypeptide ring and, on said ring, aside-chain “acylamino” group, optionally with or without one or moreother side chains.

Substance FR901379, which is a representative example of said “cycliclipopeptide compound”, is a known antifungal substance produced by themicroorganism Coleophoma sp. F-11899 (FERM BP-2635) (described inJapanese Kokai Tokkyo Koho H3-184921) and having the following chemicalformula [Ia].

The “Substance FR901379 analog” means a compound of the followinggeneral formula [I] or a salt thereof.

[wherein R¹ is acyl,

R² is hydroxy or acyloxy,

R³ is hydrogen or hydroxy,

R⁴ is hydrogen or hydroxy,

R⁵ is hydrogen or hydroxysulfonyloxy, and

R⁶ is hydrogen or carbamoyl.]

The novel cyclic lipopeptide acylase of the present invention is anacylase derived from a strain of microorganism of the genus Streptomyceswhich is capable of deacylating the side chain “acylamino” group of saidcyclic lipopeptide compound to an “amino” group. Specifically, it is anacylase which deacylates the palmitoyl side chain of substance FR901379or a salt thereof or the acyl side chain of said Substance FR901379analog of general formula [I] or a salt thereof to specifically producea compound of the following chemical formula [IIa] (Substance FR179642)or a salt thereof:

or an FR179642 analog of the following general formula [II], inclusiveof Substance FR179642), or a salt thereof.

[wherein R², R³, R⁴, R³, and R⁶ are the same groups as respectivelydefined herein before.]

The preferred salts of compounds [I] and [II] are nontoxic mono- ordi-salts of the conventional kinds. Thus, metal salts such as alkalimetal salts (e.g. sodium salt, potassium salt, etc.), alkaline earthmetal salts (e.g. calcium salt, magnesium salt, etc.), ammonium salt,salts with organic bases (e.g. trimethylamine salt, triethylamine salt,pyridine salt, picoline salt, dicyclohexylamine salt,N,N′-dibenzylethylenediamine salt, etc.), organic acid addition salts(e.g. formate, acetate, trifluoroacetate, maleate, tartarate,methanesulfonate, benzenesulfonate, toluenesulfonate, etc.), inorganicacid addition salts (e.g. hydrochloride, hydrobromide, hydroiodide,sulfate, phosphate, etc.), and salts with amino acids (e.g. arginine,aspartic acid, glutamic acid, etc.) can be mentioned.

The preferred “lower alkyl” is a straight-chain or branched alkyl groupsof 1˜6 carbon atom(s), such as methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tert-butyl, pentyl, isopentyl, and hexyl. Among them, alkylgroups of 1˜4 carbon atom(s) are preferred and methyl is particularlypreferred.

The preferred “higher alkyl” includes straight-chain or branched alkylgroups of 7˜20 carbon atom(s), such as heptyl, octyl, 3,5-dimethyloctyl,3,7-dimethyloctyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl,pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, and eicosyl.The preferred “lower alkoxy” includes straight-chain or branched groupssuch as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutyl,tert-butoxy, pentyloxy, tert-pentyloxy, neo-pentyloxy, hexyloxy, andisohexyloxy.

The preferred “higher alkoxy” includes straight-chain or branched groupssuch as heptyloxy, octyloxy, 3,5-dimethyloctyloxy, 3,7-dimethyloctyloxy,nonyloxy, decyloxy, undecyloxy, dodecyloxy, tri-decyloxy, tetradecyloxy,hexadecyloxy, heptadecyloxy, octadecyloxy, nonadecyloxy, and eicosyloxy.

The preferred “aryl” includes phenyl optionally having lower alkyl (e.g.phenyl, mesityl, tolyl, etc.), naphthyl and anthryl, and the like.

The preferred “acyl” moiety in the term of “acylamino” or “acyl” groupincludes aliphatic acyl, aromatic acyl, heterocyclic acyl,aryl-substituted aliphatic acyl, and heterocyclic-substituted aliphaticacyl derived from carboxylic acids, carbonic acids, carbamic acids, andsulfonic acids.

The preferred “acyl” includes lower alkanoyl (e.g. formyl, acetyl,propionyl, butyryl, isobutyryl, valeryl, hexanoyl, pivaloyl, etc.) whichmay have one or more (preferably 1˜3) suitable substituent(s) such asaryl (e.g. phenyl, naphthyl, anthryl, etc.) which may have one or more(preferably 1˜3) suitable substituent(s) such as halogen (e.g. fluoro,chloro, bromo, iodo, etc.), hydroxy, said higher alkoxy, said aryl,etc.; said lower alkoxy; amino; protected amino [preferably acylaminosuch as lower alkoxycarbonylamino (e.g. methoxycarbonylamino,ethoxycarbonylamino, propoxycarbonylamino, butoxycarbonylamino,tert-butoxycarbonylamino, pentyloxycarbonylamino, hexyloxycarbonylamino,etc.), etc.]; di(lower)alkylamino (e.g. dimethylamino,N-methylethylamino, diethylamino, N-propylbutylamino, dipentylamino,dihexylamino, etc.); lower alkoxyimino (e.g. methoxyimino, ethoxyimino,propoxyimino, butoxyimino, tert-butoxyimino, pentyloxyimino,hexyloxyimino, etc.); ar(lower)alkoxyimino (e.g. benzyloxyimino,phenethyloxyimino, benzhydryloxyimino, etc.) such as phenyl (lower)alkoxyimino which may have one or more (preferably 1˜3) suitablesubstituent(s) such as said higher alkoxy; heterocyclicthio (preferablypyridylthio) which may have one or more (preferably 1˜3) suitablesubstituent(s) such as higher alkyl (e.g. heptyl, octyl, 2-ethylhexyl,nonyl, decyl, 3,7-dimethyloctyl, undecyl, dodecyl, tridecyl, tetradecyl,pentadecyl, 3-methyl-10-ethyldodecyl, hexadecyl, heptadecyl, octadecyl,nonadecyl, eicosyl, etc.); andheterocyclicgroup (e.g. thienyl,imidazolyl, pyrazolyl, furyl, tetrazolyl, thiazolyl, thiadiazolyl, etc.)which may have one or more (preferably 1˜3) suitable substituent(s) suchas amino, said protected amino, said higher alkyl, and the like.;

higher alkanoyl (e.g. heptanoyl, octanoyl, nonanoyl, decanoyl,undecanoyl, lauroyl, tridecanoyl, myristoyl, pentadecanoyl, palmitoyl,10,12-dimethyltetradecanoyl, heptadecanoyl, stearoyl, nonadecanoyl,eicosanoyl, etc.);

lower alkenoyl (e.g. acryloyl, methacryloyl, crotonoyl, 3-pentenoyl,5-hexenoyl, etc.) which may have one or more (preferably 1˜3) suitablesubstituent(s) such as said aryl optionally having one or more(preferably 1˜3) suitable substituent(s) such as said higher alkoxy,etc.;

higher alkenoyl (e.g. 4-heptenoyl, 3-octenoyl, 3,6-decadienoyl,3,7,11-trimethyl-2,6,10-dodecatrienoyl, 4,10-heptadecadienoyl, etc.);

lower alkoxycarbonyl (e.g. methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, butoxycarbonyl, t-butoxycarbonyl, pentyloxycarbonyl,hexyloxycarbonyl, etc.);

higher alkoxycarbonyl (e.g. heptyloxycarbonyl, octyloxycarbonyl,2-ethylhexyloxycarbonyl, nonyloxycarbonyl, decyloxycarbonyl,3,7-dimethyloctyloxycarbonyl, undecyloxycarbonyl, dodecyloxycarbonyl,tridecyloxycarbonyl, tetradecyloxycarbonyl, pentadecyloxycarbonyl,3-methyl-10-ethyldodecyloxycarbonyl, hexadecyloxycarbonyl,heptadecyloxycarbonyl, octadecyloxycarbonyl, nonadecyloxycarbonyl,eicosyloxycarbonyl, etc.);

aryloxycarbonyl (e.g. phenoxycarbonyl, naphthyloxycarbonyl, etc.);

arylglyoxyloyl (e.g. phenylglyoxyloyl, naphthylglyoxyloyl, etc.);

ar(lower)alkoxycarbonyl which may have one or more suitablesubstituent(s), for example phenyl-(lower)alkoxycarbonyl which may havenitro or lower alkoxy (e.g. benzyloxycarbonyl, phenethyloxycarbonyl,p-nitrobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, etc.);

lower alkylsulfonyl (e.g. methylsulfonyl, ethylsulfonyl, propylsulfonyl,isopropylsulfonyl, pentylsulfonyl, butylsulfonyl, etc.);

arylsulfonyl (e.g. phenylsulfonyl, naphthylsulfonyl, etc.) which mayhave one or more (preferably 1˜3) suitable substituent(s) such as saidlower alkyl, said higher alkoxy, and the like;

ar(lower)alkylsulfonyl (e.g. benzylsulfonyl, phenethylsulfonyl,benzhydrylsulfonyl, etc.), for example phenyl (lower)alkylsulfonyl; and

aroyl (e.g. benzoyl, naphthoyl, anthrylcarbonyl, etc.) which may haveone or more (preferably 1˜5) suitable substituent(s) such as saidhalogen; lower alkyl (e.g. methyl, ethyl, propyl, butyl, tert-butyl,pentyl, hexyl, etc.); said higher alkyl; lower alkoxy (e.g. methoxy,ethoxy, propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, etc.) whichmay have one or more (preferably 1˜10) suitable substituent(s) such assaid lower alkoxy, said halogen, said aryl, and the like; higher alkoxy(e.g. heptyloxy, octyloxy, 2-ethylhexyloxy, nonyloxy, decyloxy,3,7-dimethyloctyloxy, undecyloxy, dodecyloxy, tridecyloxy,tetradecyloxy, pentadecyloxy, 3-methyl-10-ethyldodecyloxy, hexadecyloxy,heptadecyloxy, octadecyloxy, nonadecyloxy, eicosyloxy, etc.) which mayhave one or more (preferably 1˜17) suitable substituent(s) such as saidhalogen, and the like; higher alkenyloxy (e.g. 3-heptenyloxy,7-octenyloxy, 2,6-octadienyloxy, 5-nonenyloxy, 1-decenyloxy,3,7-dimethyl-6-octenyloxy, 3,7-dimethyl-2,6-octadienyloxy,8-undecenyloxy, 3,6,8-dodecatrienyloxy, 5-tridecenyloxy,7-tetradecenyloxy, 1,8-pentadecadienyloxy, 15-hexadecenyloxy,11-heptadecenyloxy, 7-octadecenyloxy, 10-nonadecenyloxy,18-eicosenyloxy, etc.); carboxy; said aryl which may have one or more(preferably 1˜3) suitable substituent(s) such as said higher alkoxy andthe like; and aryloxy (e.g. phenoxy, naphthyloxy, anthryloxy, etc.)which may have one or more (preferably 1˜3) suitable substituent(s) suchas said lower alkoxy and said higher alkoxy.

Among the above examples of “acyl”, higher alkanoyl group is preferredand palmitoyl is particularly preferred.

The preferred “acyl” moiety in the term of “acyloxy” can be referred toaforementioned “acyl” group.

The preferred “acyloxy” group includes lower alkanoyloxy (e.g.formyloxy, acetyloxy, propionyloxy, butyryloxy, isobutyryloxy,valeryloxy, hexanoyloxy, pivaloyloxy, etc.) or phosphonoxy.

The novel cyclic lipopeptide acylase of the invention can be produced bygrowing an acylase-producing strain of microorganism belonging to thegenus Streptomyces, such as Streptomyces anulatus No. 4811 (FERMBP-5808), Streptomyces anulatus No. 8703 (FERM BP-5810), or Streptomycessp. No. 6907 (FERM BP-5809), in a culture medium.

Generally, this novel acylase can be produced by growing said novelacylase-producing strain of microorganism in an aqueous mediumcontaining assimilable carbon and digestable nitrogen sources preferablyunder aerobic conditions, for example by shake culture and submergedculture.

The preferred carbon source for the medium includes variouscarbohydrates such as glucose, xylose, galactose, glycerin, starch,dextrin, and the like. As other carbon sources, maltose, rhamnose,raffinose, arabinose, mannose, salicin, sodium succinate, and the likecan be mentioned.

The preferred nitrogen source includes yeast extract, peptone, glutenmeal, cottonseed flour, soybean flour, corn steep liquor, dried yeast,wheat germs, down meal, peanut flour, and the like. as well as inorganicor organic nitrogenous compounds such as ammonium salts, (e.g. ammoniumnitrate, ammonium sulfate, ammonium phosphate, etc.), urea, and aminoacids.

While those carbon and nitrogen sources are used preferably in suitablecombinations, even materials of low purity can be used provided thatthey contain suitable amounts of growth factors and reasonable amountsof inorganic nutrients and it is not always necessary to use them in thepure form. Optionally, the medium may be supplemented with sodiumcarbonate or potassium carbonate, sodium phosphate or potassiumphosphate, sodium chloride or potassium chloride, sodium iodide orpotassium iodide, and inorganic salts such as magnesium salts, coppersalts and cobalt salts. Particularly, when the culture medium produces acopious foam, a deforming agent such as liquidparaffin, fatty oil,vegetable oil, mineral oil, and silicone may be added as necessary.

For the mass production of the novel acylase, the submerged aerobiccultural method is preferred. For minor-scale production, shake cultureor surface culture is carried out in a flask or bottle. Forlarge-capacity tank culture, the fermentation tank is preferablyinoculated with a seed culture for avoiding a delay in growth in theproduction line for the novel acylase. Thus, preferably a comparativelysmall amount of culture medium is first inoculated with the spores ormycelium of the strain and incubated to prepare a seed culture which isthen aseptically transferred to a large-capacity fermentation tank. Themedium for this seed culture may be substantially the same as productionmedium for the novel acylase or different from the production medium.

The agitation and aeration of the fermentation system can be carried outin various ways. For example, the agitation can be effected by using apropeller or other similar stirring device, rotating or shaking thefermentator, by means of a pump of choice, or passing sterile airthrough the medium. The aeration can be achieved by blowing sterile airinto the fermentation system.

The fermentation is carried out generally within the temperature rangeof about 20˜32° C., preferably 25˜30° C., and the pH range of 6˜8 forabout 50˜150 hours. Those conditions may be modified according to othercultural conditions and fermentation scale.

The novel acylase thus produced can be recovered from the fermentationbroth by techniques which are routinely used for recovery of other knownbioactive substances. The novel acylase elaborated is found in both thegrown mycelium and the filtrate. Therefore, the novel acylase can beseparated from the mycelial cake and filtrate available upon filtrationor centrifugation of the broth and purified by the conventional methodssuch as concentration under reduced pressure, freeze-drying, extractionwith the common solvent, pH adjustment, treatment with a conventionalresin, (e.g. anion exchange resin, cation exchange resin, nonionicadsorbent resin, etc.), treatment with an ordinary adsorbent such asactive charcoal, silicic acid, silica gel, cellulose, alumina,crystallization, and recrystallization.

The following examples illustrate the acylase produced by Streptomycesanulatus No. 4811, Streptomyces anulatus No. 8703, or Streptomyces sp.No. 6907 in further detail and should by no means be construed asdefining the scope of the invention.

EXAMPLE 1-1 Production of the Acylase of the Streptomyces Anulatus No.4811 Origin

A conical flask of 100-ml capacity was charged with 30 ml of a seedculture medium containing 1% of corn starch, 1% of glucose, 0.5% ofpeanut flour, 0.5% of soybean flour, 0.5% of dried yeast, and 0.2% ofcalcium carbonate and sterilized at 120° C. for 20 minutes. Thesterilized flask was then inoculated with 1˜2 loopful(s) of a slant agarculture of Streptomyces anulatus No. 4811 and incubated under shaking at30° C. for 3 days to provide a seed culture.

Meanwhile, a production medium containing 4% of sucrose, 1% of peanutflour, 1% of dried yeast, 0.05% of potassium dihydrogen phosphate, 0.12%of dipotassium hydrogen phosphate, and 0.025% of magnesium sulfate 7H₂Owas adjusted to pH 6.5 and a conical flask of 500-ml capacity was filledwith 100 ml of the production medium and sterilized at 120° C. for 20minutes. The sterilized flask was inoculated with 2 ml of the above seedculture and incubated under shaking at 30° C. for 3 days to provide afermentation broth.

EXAMPLE 1-2 Production of the Acylase of the Streptomyces Anulatus No.8703 Origin

A conical flask of 100-ml capacity was charged with 30 ml of a seedculture medium containing 1% of corn starch, 0.1% of glucose, 0.5% ofpeanut flour, 0.5% of soybean flour, 0.5% of dried yeast, and 0.2% ofcalcium carbonate and sterilized at 120° C. for 20 minutes. Thesterilized flask was then inoculated with 1˜2 loopful(s) of a slant agarculture of Streptomyces anulatus No. 8703 and incubated under shaking at30° C. for 3 days to provide a seed culture.

Meanwhile, a production medium containing 4% of sucrose, 1% of peanutflour, 1% of dried yeast, 0.05% of potassium dihydrogen phosphate, 0.12%of dipotassium hydrogen phosphate, and 0.025% of magnesium sulfate 7H₂Owas adjusted to pH 6.5 and a conical flask of 500-ml capacity was filledwith 100 ml of the production medium and sterilized at 120° C. for 20minutes. The sterilized flask was inoculated with 2 ml of the above seedculture and incubated under shaking at 30° C. for 3 days to provide afermentation broth.

EXAMPLE 1-3 Production of the Acylase of the Strertomyces sp. No. 6907Origin

A conical flask of 100-ml capacity was charged with 30 ml of a seedmedium containing 6% of soluble starch, 4% of defatted soybean meal, and0.5% of calcium carbonate and sterilized at 120° C. for 20 minutes. Thesterilized flask was then inoculated with 1[2 loopful(s) of a slant agarculture of Streptomyces sp. No. 6907 and incubated under shaking at 30°C. for 3 days to provide a seed culture.

Meanwhile, a production medium containing 4% of sucrose, 1% of peanutflour, 1% of dried yeast, and 0.5% of calcium carbonate was adjusted topH 6.5 and a conical flask of 500-ml capacity was charged with 100 ml ofthe production medium and sterilized at 120° C. for 20 minutes Thesterilized flask was inoculated with 2 ml of the above seed culture andincubated under shaking at 30° C. for 4 days to provide a fermentationbroth.

EXAMPLE 1-4 Production of the Acylase of the Allotted StreptomycesOrigin

Using the following Streptomyces strain allotted from Institute forFermentation, Osaka (IFO, 2-17-85, Juso Hommachi, Yodogawa-ku,Osaka-shi), the cultivation procedure of Example 1-1 for Streptomycesanulatus No. 4811 was otherwise repeated to provide a fermentationbroth.

Streptomyces griseus subsp. griseus IFO 13189

The method of deacylating the acyl side chain of the antifungal cycliclipopeptide compound (e.g. FR901379 or its analog) with the novel cycliclipopeptide acylase of the invention is now described in detail.

The deacylation method of the invention can be carried out in thefollowing manner.

A suitable production medium is inoculated with a strain ofmicroorganism belonging to the genus Streptomyces and capable ofproducing the novel acylase and the inoculated medium was incubated atabout 25˜35° C. for a few days to provide a fermentation broth. Thisfermentation broth is added to the substrate cyclic lipopeptidecompound, such as Substance FR901379, and the mixture is incubated at45˜60° C. and pH about 6.0˜9. Then, the cyclic peptide such as SubstanceFR179642 is detected and separated by high-performance liquidchromatography(HPLC).

The following examples are intended to illustrate the deacylation methodof the invention in detail and should by no means be construed asdefining the scope of the invention.

EXAMPLE 2-1

To 700 μl of the fermentation broth of Streptomyces anulatus No. 4811obtained in Example 1-1 was added 100 μl of an aqueous solution ofSubstance FR901379 (100 mg/ml) (10 mg as Substance FR901379; 8.35 μmol)together with 100 μl of methanol and 100 μl of buffer (0.5 M potassiumdihydrogen phosphate-disodium hydrogen phosphate buffer; pH 6.0), andthe reaction was carried out at 30° C. for 30 minutes. The reaction wasthen stopped with 1 ml of 4% acetic acid and following addition of 2 mlof methanol, the mixture was filtered through a membrane filter (0.45μm) to remove the macromolecular protein and other fraction andsubjected to HPLC and the acylase activity of the skeletal substanceFR179642 produced was monitored and assayed at 210 nm.

Using a variable wavelength UV detector (Shimadzu SPD-10A), a pump(Shimadzu LC-10AD) and an integrator (Shimadzu C-R6A) as theinstrumentation, LiChrospher 100RP-18(e) (250 mm×4 mm i.d., particlediameter 5 μm(E.Merck)) as the stationary phase, and 3%acetonitrile/0.5% ammonium dihydrogen phosphate as the mobile phase,Substance FR179642 was eluted at a flow rate of 1 ml/min. The retentiontime of Substance FR179642 was about 6.3 minutes. The yield of SubstanceFR179642 as calculated from HPLC data was 730 μg (0.78 μmol).

EXAMPLE 2-2

To 700 μl of the fermentation broth of Streptomyces anulatus No. 8703obtained in Example 1-2 was added 100 μl of an aqueous solution ofSubstance FR901379 (100 mg/ml) (10 mg as Substance FR901379; 8.35 μmol)together with 100 μl of methanol and 100 μl of buffer (0.5 M potassiumdihydrogen phosphate-disodium hydrogen phosphate buffer; pH 6.0), andthe reaction was carried out at 30° C. for 30 minutes. The reaction wasthen stopped with 1 ml of 4% acetic acid and following addition of 2 mlof methanol, the mixture was filtered through a membrane filter (0.45μm) to remove the macromolecular protein and other fraction andsubjected to HPLC and the acylase activity of the skeletal substanceFR179642 produced was monitored and assayed at 210 nm.

Using a variable wavelength UV detector (Shimadzu SPD-10A), a pump(Shimadzu LC-10AD) and an integrator (Shimadzu C-R6A) as theinstrumentation, LiChrospher 100RP-18(e) (250 mm×4 mm i.d., particlediameter 5 μm(E.Merck)) as the stationary phase, and 3%acetonitrile/0.5% ammonium dihydrogen phosphate as the mobile phase,Substance FR179642 was eluted at a flow rate of 1 ml/min. The retentiontime of Substance FR179642 was about 6.3 minutes. The yield of SubstanceFR179642 as calculated from HPLC data was 830 μg (0.89 μmol).

EXAMPLE 2-3

To 700 μl of the fermentation broth of Streptomyces sp. No. 6907obtained in Example 1-3 was added 100 μl of an aqueous solution ofSubstance FR901379 (100 mg/ml) (10 mg as Substance FR901379; 8.35 μmol)together with 100 μl of methanol and 100 μl of buffer (0.5 M potassiumdihydrogen phosphate-disodium hydrogen phosphate buffer; pH 6.0), andthe reaction was carried out at 30° C. for 30 minutes. The reaction wasthen stopped with 4% acetic acid and following addition of 2 ml ofmethanol, the mixture was filtered through a membrane filter (0.45 μm)to remove the macromolecular protein and other fraction and subjected toHPLC and the acylase activity of the skeletal substance FR179642produced was monitored and assayed at 210 nm.

Using a variable wavelength UV detector (Shimadzu SPD-10A), a pump(Shimadzu LC-10AD) and an integrator (Shimadzu C-R6A) as theinstrumentation, LiChrospher 100RP-18(e) (250 mm×4 mm i.d., particlediameter 5 μm(E.Merck)) as the stationary phase, and 3%acetonitrile/0.5% ammonium dihydrogen phosphate as the mobile phase,Substance FR179642 was eluted at a flow rate of 1 ml/min. The retentiontime of Substance FR179642 was about 6.3 minutes. The yield of SubstanceFR179642 as calculated from HPLC data was 560 μg (0.60 μmol). The Kmvalue determined by the Lineweaver-Burk method was 257 μM and Vmax was14.3 U/mg-protein.

EXAMPLE 2-4

To 100 μl of the fermentation broth of Streptomyces sp. No. 6907obtained in Example 1-3 was added 100 μl of a dimethyl sulfoxidesolution of Aculeacin A (100 mg/ml) (10 mg as Aculeacin A; 9.65 μmol)together with 500 μl of 1.2 M KCl-containing 500 mM potassium dihydrogenphosphate-disodium hydrogen phosphate buffer (pH 7.0) and 300 μl ofwater, and the reaction was carried out at 40° C. for 15 minutes. Thereaction was then stopped with 1 ml of 4% acetic acid and followingaddition of 2 ml of methanol, the mixture was filtered through amembrane filter (0.45 μm) to remove the macromolecular protein and otherfraction and subjected to HPLC and the acylase activity of the nuclearsubstance Aculeacin A produced was monitored at 210 nm and determined.Using a variable wavelength UV detector (Shimadzu SPD-10A), a pump(Shimadzu LC-10AD) and an integrator (Shimadzu C-R6A) as theinstrumentation, LiChrospher 100RP-18(e) (250 mm×4 mm i.d., particlediameter 5 μm (E.Merck)) as the stationary phase, and 4% acetonitrile/1%ammonium dihydrogen phosphate as the mobile phase, nuclear substanceAculeacin A was eluted at a flow rate of 1 ml/min. The retention time ofnuclear substance Aculeacin A was about 8.7 minutes. The yield ofnuclear substance Aculeacin A as calculated from HPLC data was 370 μg(0.47 μmol). The Km value determined by the Lineweaver-Burk method was279 μM and Vmax was 16.8 U/mg-protein.

EXAMPLE 2-5

To 100 μl of the fermentation broth of Streptomyces sp. No. 6907obtained in Example 1-3 was added 100 μl of a dimethyl sulfoxidesolution of Echinocandin B (100 mg/ml) (10 mg as Echinocandin B; 9.43μmol) together with 500 μl of 1.2 M KCl-containing 500 mM potassiumdihydrogen phosphate-disodium hydrogen phosphate buffer (pH 7.0) and 300μl of water, and the reaction was carried out at 40° C. for 15 minutes.The reaction was then stopped with 1 ml of 4% acetic acid and followingaddition of 2 ml of methanol, the mixture was filtered through amembrane filter (0.45 μm) to remove the macromolecular protein and otherfraction and subjected to HPLC and the acylase activity of the nuclearsubstance Echinocandin B produced was monitored and assayed at 210 nm.Using a variable wavelength UV detector (Shimadzu SPD-10A), a pump(Shimadzu LC-10AD) and an integrator (Shimadzu C-R6A) as theinstrumentation, LiChrospher 100RP-18(e) (250 mm×4 mm i.d., particlediameter 5 μm (E.Merck)) as the stationary phase, and 4% acetonitrile/1%ammonium dihydrogen phosphate as the mobile phase, nuclear substanceEchinocandin B was eluted at a flow rate of 1 ml/min. The retention timeof nuclear substance Echinocandin B was about 8.7 minutes. The yield ofnuclear substance Echinocandin B as calculated from HPLC data was 90 μg(0.11 μmol). The Km value determined by the Lineweaver-Burk method was146 μM and Vmax was 7.85 U/mg-protein.

The following characterizes the process of deacylation of the acyl sidechain of an antifungal cyclic lipopeptide compound (e.g. SubstanceFR901379) by the acylase derived from the novel acylase-producing strainof microorganism belonging to the genus Streptomyces in accordance withthe present invention.

The data shown were generated by the experimental method described inExample 2-1, Example 2-2, or Example 2-3 but varying the kind of buffer(0.5 M sodium citrate buffer, 0.5 M potassium dihydrogenphosphate-disodium hydrogen phosphate buffer, and Tris-HCl buffer wereused in various combinations), reaction temperature, and addition levelof methanol. Each acylase activity is shown in the concentration(measured by HPLC) of Substance FR179642 at completion of the reaction.

Optimal Reaction pH

Test 1-1

Optimal pH for the acylase produced by Streptomyces anulatus No. 4811

Concentration of Substance FR179642 pH at completion of reaction (μg/ml)3  0 4  90 5 440 6 630 6.5 710 7 770 8 810 9 740

Test 1-2

Optimal pH for the acylase produced by Streptomyces anulatus No. 8703

Concentration of Substance FR179642 pH at completion of reaction (μg/ml)3  0 4 240 5 590 6 810 7 1,070   8 1,300   9 1,270  

Test 1-3

Optimal pH for the acylase produced by Streptomyces sp. No. 6907

Concentration of Substance FR179642 pH at completion of reaction (μg/ml)3  0 4 190 5 280 6 300 7 470 8 800 9 1,000  

The above results indicate that, in the working of the invention withany of the above acylases, the reaction proceeds at and over weakacidity (pH about 4) and that the reaction rate is increased as the pHis enhanced.

Optimal Reaction Temperature

Test 2-1

Optimal temperature for the acylase produced by Streptomyces anulatusNo. 4811

Tempera- Concentration of Substance FR179642 ture at completion ofreaction (μg/ml) 25   360 30   630 40 1,490 50 3,120 60 1,110 70   60

Test 2-2

Optimal temperature for the acylase produced by Streptomyces anulatusNo. 8703

Tempera- Concentration of Substance FR179642 ture at completion ofreaction (μg/ml) 25   410 30   770 40 1,760 50 3,160 60 2,110 70   120

Test 2-3

Optimal temperature for the acylase produced by Streptomyces sp. No.6907

Tempera- Concentration of Substance FR179642 ture at completion ofreaction (μg/ml) 25   600 30   710 40 3,010 50 4,620 60 1,660 70   130

The above results indicate that, in the working of the invention withany of the above acylases, the optimal reaction temperature is 40˜60° C.

Effect of Methanol Added to the Reaction System

Test Example 3-1

The effect of methanol on the reaction using the acylase produced byStreptomyces anulatus No. 4811

Concentration Concentration of Substance FR179642 of methanol (%) atcompletion of reaction (μg/ml)  0 390  5 570 10 630 15 590 20 550 30 39040 130 50  10

Test 3-2

The effect of methanol on the reaction using the acylase produced byStreptomyces anulatus No. 8703

Concentration Concentration of Substance FR179642 of methanol (%) atcompletion of reaction (μg/ml)  0 320  5 580 10 770 15 750 20 660 30 36040 140 50  50

Test 3-3

The effect of methanol on the reaction using the acylase produced byStreptomyces sp. No. 6907

Concentration Concentration of Substance FR179642 of methanol (%) atcompletion of reaction (μg/ml)  0 330  5 580 10 710 15 630 20 530 30 14040 130 50  90

The above results indicate that, in the working of the invention withany of the above acylases, the activity is increased 1.6-fold through2.2-fold in the presence of 5˜20% of methanol.

The cyclic lipopeptide acylase obtained by growing an acylase-producingstrain of microorganism of the genus Streptomyces is now described indetail.

Characteristics of the Acylase Produced by Streptomyces sp. 6907

1) Activity:

This enzyme catalyzes the deacylation of the lipid acyl moiety of thecyclic lipopeptide compound represented by Substance FR901379 andFR901379 analogs such as Echinocandin B and Aculeacin A.

2) Optimal pH: pH 8˜9

3) Optimal temperature for activity: about 50° C.

4) Inhibition, activation, and stabilization: Methanol: The enzyme isactivated concentration-dependently up to 10% in the reaction mixtureand is inhibited at higher concentrations.

5) Molecular weight: SDS-PAGE of the purified enzyme gave two bands.

Large peptide (SEQ ID NO:1): 61 kD

Small peptide: 19 kD

6) Crystal structure:

The purified protein was small in amount and not crystalline.

7) Amino acid analysis: N-terminal amino acid sequences

Large Peptide:

Ser-Asn-Ala-Val-Ala-Phe-Asp-Gly-Ser-Thr-Thr-Val-Asn-Gly-Arg-Gly-Leu-Leu-Leu-Gly-. . .

Small Peptide (SEQ ID NO:2):

Gly-Ser-Gly-Leu-Ser-Ala-Val-Ile-Arg-Tyr-Thr-Glu-Tyr-Gly-Ile-Pro-His-Ile-Val-Ala-. . .

8) Substrate specificity:

The enzyme acts as a catalyst for FR901379, Echinocandin B and AculeacinA. However, it does not act upon FR901469.

The following examples illustrate the acylase of the invention infurther detail but should by no means be construed as defining the scopeof the invention.

Purification of The Acylase:

EXAMPLE 3

The fermentation broth of Streptomyces sp. No. 6907 obtained in Example1˜3 was extracted with 1.5 M KCl under stirring at a low temperature andthe filtrate separated with No. 2 filter paper was desalted with an UFmembrane (Asahi Chemical Industries; AIP-1010) [20 mM Tris-HCl buffer(pH 9) substituted] and passed through an HP-20 column. The effluent wasapplied to a DEAE-Toyopearl column (Cl⁻-form) and elution was carriedout with 0.3 M NaCl-supplemented 20 mM Tris-HCl buffer (pH 9). In theresulting active fraction, a 0.5 M equivalent of (NH₄)₂SO₄ wasdissolved, and the solution was applied to a phenyl-Toyopearl column,elution being then carried out with 0.1 M NaCl-supplemented 50 mMTris-HCl buffer (pH 8). The resulting active fraction was desalted andconcentrated with a DF membrane (Asahi Chemical Industries; SIP-0013)[20 mM Tris-HCl buffer (pH 9) substituted] and subjected to gelpermeation chromatography on a YMC-Diol column (mobile phase: 0.2 MNaCl-0.1 M potassium dihydrogen phosphate-disodium hydrogen phosphatebuffer pH 7.0). The active fraction was further purified by preparativechromatography on a reversed phase Cosmosil 5C4-AR-300 column [mobilephase: (A solution) 0.5% trifluoroacetic acid, (B solution) 0.5%trifluoroacetic acid-80% acetonitrile, A:B=60:40→40:60 (lineargradient)], whereby two bands were obtained. The thus-purified acylasewas subjected to SDS-PAGE in this manner, two different peptides havingmolecular masses of 61 kD and 19 kD, respectively, were provided.

Substrate Specificity

Substance FR901469 is a known substance (described in WO 92/19648)having antifungal activity as produced by the fungal strain No. 11243(FERM BP-3373). It is a compound of the following chemical formula[III]:

The Actinoplanes utahensis-derived acylase (described in Japanese KokaiTokkyo Koho H4-228072), which has deacylation activity as does the novelacylase according to the present invention, exerts a catalytic action onsaid Substance FR901469 and its salt to produce Substance FR181131(described in WO 96/30399) of the following chemical formula [IV]:

In contrast, the novel acylase of the present invention exerts nocatalytic action on said Substance FR901469 and its salt.

Examples of the invention are presented below.

EXAMPLE 4-1

To 20 ml of a fermentation broth of Actinoplanes utahensis IF013244 asprepared by the procedure described in J. Antibiotics, Vol. 41, p.1085-1092 ('88) was added 1 ml of an aqueous solution of SubstanceFR901469 (200 mg/ml) (0.2 g as Substance FR901469; 130 μmol) togetherwith 2.9 g of disodium hydrogen phosphate and 60 ml of water, and thereaction was carried out at 60° C. for 24 hours. After completion of thereaction, the reaction mixture was filtered through a membrane filter(0.45 μm) to remove the high molecular protein and other fraction andpurified by HPLC under monitoring for Substance FR181131 at 210 nm andthe acylase activity was determined. Using a variable wavelength UVdetector (Shimadzu SPD-10A), a pump (Shimadzu LC-10AD) and an integrator(Shimadzu C-R6A) as the instrumentation, YMC AM303 (250 mm×4 mm i.d.,particle diameter 5 μm) as the stationary phase, and 12.5%acetonitrile/0.5% ammonium dihydrogen phosphate as the mobile phase,Substance FR181131 was eluted at a flow rate of 1 ml/min. The retentiontime of Substance FR181131 was about 7.3 minutes. The yield of SubstanceFR181131 as calculated from HPLC data was 80 mg (68 μmol).

EXAMPLE 4-2

To 2.0 ml of the fermentation broth of Streptomyces sp. No. 6907obtained in Example 1-3 was added 1 ml of an aqueous solution ofSubstance FR901469 (200 mg/ml) (0.2 g as Substance FR901469; 130 μmol)together with 2.9 g of disodium hydrogen phosphate and 60 ml of water,and the reaction was carried out at 60° C. for 24 hours. Aftercompletion of the reaction, the reaction mixture was filtered through amembrane filter (0.45 μm) to remove the high molecular protein and otherfraction and purified by HPLC under monitoring for Substance FR181131 at210 nm and the acylase activity was determined. Using a variablewavelength UV detector (Shimadzu SPD-10A), a pump (Shimadzu LC-10AD) andan integrator (Shimadzu C-R6A) as the instrumentation, YMC AM303 (250mm×4 mm i.d., particle diameter 5 μm) as the stationary phase, and 12.5%acetonitrile/0.5% ammonium dihydrogen phosphate as the mobile phase,Substance FR181131 was eluted at a flow rate of 1 ml/min. The retentiontime of Substance FR181131 was about 7.3 minutes. The yield of SubstanceFR181131 as calculated from HPLC data was not over 2 mg (1.7 μmol)(below detection limit).

Coleophoma sp. F-11899, which produces Substance FR901379, andStreptomyces anulatus No. 4811, Streptomyces anulatus No. 8703, andStreptomyces sp. No. 6907, all of which produce the acylase of theinvention, have all been deposited with National Institute of Bioscienceand Human Technology (NIBH, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki,Japan).

Microorganism Accession No. Coleophoma sp. F-11899 FERM BP-2635Streptomyces anulatus No. 4811 FERM BP-5808 Streptomyces anulatus No.8703 FERM BP-5810 Streptomyces sp. No. 6907 FERM BP-5809 Strain No.11243 FERM BP-3373

2 1 20 PRT Streptomyces sp.6907 1 Ser Asn Ala Val Ala Phe Asp Gly SerThr Thr Val Asn Gly Arg Gly 1 5 10 15 Leu Leu Leu Gly 20 2 20 PRTStreptomyces sp.6907 2 Gly Ser Gly Leu Ser Ala Val Ile Arg Tyr Thr GluTyr Gly Ile Pro 1 5 10 15 His Ile Val Ala 20

What is claimed is:
 1. A biologically pure culture of Streptomycesanulatus 4811 (FERM BP-5808).
 2. A method of producing an acylase fromthe biologically pure culture of Streptomyces anulatus of claim 1,comprising culturing the biologically pure culture of Streptomycesanulatus in a medium under conditions and for a time sufficient toproduce the acylase; and collecting the acylase, wherein the acylasecatalyzes the deacylation of the acyl group R¹ of a cyclic lipopeptidecompound of the following general formula I or a salt thereof:

wherein R¹ is acyl; R² is hydroxy or acyloxy; R³ is hydrogen or hydroxy;R⁴ is hydrogen or hydroxy; R⁵ is hydrogen or hydroxysulfonyloxy; and R⁶is hydrogen or carbamoyl;  to produce a cyclic peptide compound of thefollowing general formula II or a salt thereof:

wherein R², R³, R⁴, R⁵, and R⁶ are as defined above; wherein said enzymedeacylates at an optimal pH of 8.0 to 9.0, at an optimal temperature of50° C., and is activated in a concentration-dependent manner bymethanol, wherein activation increases up to 10% by volume concentrationof methanol.
 3. The method of claim 2, wherein R⁵ is hydroxysulfonyloxyand R⁶ is carbamoyl.
 4. The method of claim 2, wherein said cycliclipopeptide compound is selected from the group consisting of FR901379,Echinocandin B, and Aculeacin A.
 5. The method of claim 2, wherein theacylase does not deacylate the compound FR901469.
 6. The method of claim2, wherein the acylase catalyzes the deacylation of the lipid acylmoiety of a cyclic lipopeptide at an optimal pH of 8.0.
 7. Abiologically pure culture of Streptomyces anulatus 8703 (FERM BP-5810).8. A method of producing an acylase from the biologically pure cultureof Streptomyces anulatus of claim 7, comprising culturing thebiologically pure culture of Streptomyces anulatus in a medium underconditions and for a time sufficient to produce the acylase; andcollecting the acylase, wherein the acylase catalyzes the deacylation ofthe acyl group R¹ of a cyclic lipopeptide compound of the followinggeneral formula I or a salt thereof:

wherein R¹ is acyl; R² is hydroxy or acyloxy; R³ is hydrogen or hydroxy;R⁴ is hydrogen or hydroxy; R⁵ is hydrogen or hydroxysulfonyloxy; and R⁶is hydrogen or carbamoyl;  to produce a cyclic peptide compound of thefollowing general formula II or a salt thereof:

wherein R², R³, R⁴, R⁵, and R⁶ are as defined above; wherein said enzymedeacylates at an optimal pH of 8.0 to 9.0, at an optimal temperature of50° C., and is activated in a concentration-dependent manner bymethanol, wherein activation increases up to 10% by volume concentrationmethanol.
 9. The method of claim 8, wherein R⁵ is hydroxysulfonyloxy andR⁶ is carbamoyl.
 10. The method of claim 8, wherein said cycliclipopeptide compound is selected from the group consisting of FR901379,Echinocandin B, and Aculeacin A.
 11. The method of claim 8, wherein theacylase does not deacylate the compound FR901469.
 12. The method ofclaim 8, wherein the acylase catalyzes the deacylation of the lipid acylmoiety of a cyclic lipopeptide at an optimal pH of 8.0.
 13. Abiologically pure culture of Streptomyces sp. 6907 (FERM BP-5809).
 14. Amethod of producing an acylase from the biologically pure culture ofStreptomyces sp. of claim 13, comprising culturing the biologically pureculture of Streptomyces sp. in a medium under conditions and for a timesufficient to produce the acylase; and collecting the acylase.
 15. Themethod of claim 14, herein the acylase catalyzes the deacylation of theacyl group R¹ of a cyclic lipopeptide compound of the following generalformula I or a salt thereof:

wherein R¹ is acyl; R² is hydroxy or acyloxy; R³ is hydrogen or hydroxy;R⁴ is hydrogen or hydroxy; R⁵ is hydrogen or hydroxysulfonyloxy; and R⁶is hydrogen or carbamoyl;  to produce a cyclic peptide compound of thefollowing general formula II or a salt thereof:

wherein R², R³, R⁴, R⁵, and R⁶ are as defined above; wherein said enzymedeacylates at an optimal pH of 8.0 to 9.0, at an optimal temperature of50° C., and is activated in a concentration-dependent manner bymethanol, wherein activation increases up to 10% by volume concentrationof methanol.
 16. The method of claim 15, wherein R⁵ ishydroxysulfonyloxy and R⁶ is carbamoyl.
 17. The method of claim 15,wherein said cyclic lipopeptide compound is selected from the groupconsisting of FR901379, Echinocandin B, and Aculeacin A.
 18. The methodof claim 15, wherein the acylase does not deacylate the compoundFR901469.
 19. The method of claim 15, wherein the acylase comprises alarge peptide and a small peptide, wherein said large peptide comprisesSEQ ID NO:1 and said small peptide comprises SEQ ID NO:2.
 20. The methodof claim 19, wherein said large peptide is approximately 61 kD and saidsmall peptide is approximately 19 kD as determined by SDS-PAGE.
 21. Themethod of claim 15, wherein the acylase catalyzes the deacylation of thelipid acyl moiety of a cyclic lipopeptide at an optimal pH of 9.0.